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Xenopus laevis is among the few species that are capable of fully regenerating a lost lens de novo. This occurs upon removal of the lens, when secreted factors from the retina are permitted to reach the corneaepithelium and trigger it to form a new lens. Although many studies have investigated the retinal factors that initiate lens regeneration, relatively little is known about what factors support this process and make the cornea competent to form a lens. We presently investigate the role of Retinoic acid (RA) signaling in lens regeneration in Xenopus. RA is a highly important morphogen during vertebrate development, including the development of various eye tissues, and has been previously implicated in several regenerative processes as well. For instance, Wolffian lens regeneration in the newt requires active RA signaling. In contrast, we provide evidence here that lens regeneration in Xenopus actually depends on the attenuation of RA signaling, which is regulated by the RA-degrading enzyme CYP26. Using RT-PCR we examined the expression of RA synthesis and metabolism related genes within ocular tissues. We found expression of aldh1a1, aldh1a2, and aldh1a3, as well as cyp26a1 and cyp26b1 in both normal and regenerating corneal tissue. On the other hand, cyp26c1 does not appear to be expressed in either control or regenerating corneas, but it is expressed in the lens. Additionally in the lens, we found expression of aldh1a1 and aldh1a2, but not aldh1a3. Using an inhibitor of CYP26, and separately using exogenous retinoids, as well as RA signaling inhibitors, we demonstrate that CYP26 activity is necessary for lens regeneration to occur. We also find using phosphorylated Histone H3 labeling that CYP26 antagonism reduces cell proliferation in the cornea, and using qPCR we find that exogenous retinoids alter the expression of putative corneal stem cell markers. Furthermore, the Xenopus cornea is composed of an outer layer and inner basal epithelium, as well as a deeper fibrillar layer sparsely populated with cells. We employed antibody staining to visualize the localization of CYP26A, CYP26B, and RALDH1 within these corneal layers. Immunohistochemical staining of these enzymes revealed that all 3 proteins are expressed in both the outer and basal layers. CYP26A appears to be unique in also being present in the deeper fibrillar layer, which may contain cornea stem cells. This study reveals a clear molecular difference between newt and Xenopus lens regeneration, and it implicates CYP26 in the latter regenerative process.
Fig. 1. Expression of RA signaling related genes. (A) Expression of RA-signaling related genes in control corneas ("C") and pooled regenerating corneas from 1, 3, and 5 days post-lentectomy ("R"). â+RTâ group indicates reactions that employed reverse transcriptase for generation of cDNA. ââRTâ group indicates reactions that used water in place of reverse transcriptase, as a control for genomic contamination. â+â indicates a positive control reaction performed using embryonic RNA. (B) Expression of genes in control lenses.
Fig. 2. RA signaling inhibits lens regeneration, and RA signaling antagonism does not inhibit lens regeneration. (A) Liarozole and TTNPB are potent inhibitors of cornea-lens regeneration. A high concentration of RA very strongly inhibits lens regeneration. ⎠indicates p<0.0001, using two-tailed Fischer's exact test. (B) A drug-treatment validation assay using qPCR shows that all of the drugs strongly activate RA signaling, as seen by a profound upregulation of cyp26a1, a marker of active RA signaling, in the cornea. ⎠indicates p<0.01, using unpaired t test. (C) Citral and LE-135, inhibitors of RA signaling, fail to inhibit cornea-lens regeneration. (D) A drug-treatment validation assay using qPCR shows that LE-135 attenuates expression of cyp26a1 in the cornea. ⎠indicates p<0.0001, using unpaired t test. All error bars indicate standard error.
Fig. 3. Lens regeneration assays. Representative examples are shown of positive cases of lens regeneration for each condition tested, with the exception of 20 µM RA, for which no lenses regenerated. Panels (A, E, I, M, Q, U, C, G, K, O, S, and W) are images taken with a DIC microscope. Panels (B, F, J, N, R, V, D, H, L, P, T, and X) are images of fluorescently labeled lenses with an anti-lens antibody (red). Arrowheads indicate the regenerated lens. Scale bar in X=100 µM.
Fig. 4. The effect of CYP26 inhibition, and exogenous retinoids on cell proliferation in the cornea. Nuclei are labeled with Hoechst (blue) and mitotic cells are labeled with anti-phospho-Histone H3 antibody (green). When compared to treatment with DMSO in vitro (A), treatment with TTNPB (C), 1 µM RA (D), or 20 µM RA (E) has no effect on the number of mitotic cells at in the cornea. Treatment with Liarozole (B) reduced cell division. White arrowheads indicate examples of mitotic figures found in the cornea. Note that not every mitotic figure is pointed out in (A) and (E). The white dotted outline demarcates the cornea proper from pericorneal epithelium. The results are quantified in (F). Error bars show standard error. ⎠indicates p<0.0001 using unpaired t-test. Scale bar in E=50 µm.
Fig. 5. qPCR of putative corneal stem cell markers. Corneas cultured in vitro in the presence of retinoids show upregulation of sox2, and downregulation of oct60 and p63. CYP26 inhibition with Liarozole does not affect the expression of any of these genes. Error bars show standard error. ⎠indicates p<0.05 and âŽâŽ indicates p< 0.001 using unpaired t-test.
Fig. 6. Cellular localization of CYP26 and RALDH enzymes in the cornea. Corneal whole-mount immunofluorescence staining was used to visualize the expression of CYP26A (A–C), CYP26B (D–F), and RALDH1 (G–I), in each of the 3 layers of the larval cornea—the outer epithelium (A, D, G), the basal layer (B, E, H), and the deeper fibrillar layer (C, F, I). CYP26A (green) is expressed in all layers, including the sparse cells of the fibrillar layer (arrowheads). CYP26B (green) and RALDH1 (green) are only expressed in the outer epithelial and basal layers, and are not seen in the cells of the fibrillar layer (arrowheads). DAPI (blue), phalloidin (red). Scale bar in G=20 µm.
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